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Apr 6, 2017 - Department of Macromolecular Science, Graduate School of Science, Osaka .... chromatography (SEC) was used to determine the degree of...
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Strategic Construction of Chiral Helices: Expanded Poly(L‑leucine) Containing p‑Phenylene Moieties Taka-aki Okamura* and Shuichiro Seno Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan S Supporting Information *

ABSTRACT: A series of expanded oligo- and poly(L-leucine)s, with an alternating arrangement of p-phenylene moieties and Lleucine residues, were synthesized by stepwise elongation or polycondensation and characterized by 1H NMR, SEC, ESI-MS, UV, and CD spectra. The degree of polymerization was found to be about 19 by SEC. Each monomer unit, i.e., expanded Lleucine, behaved like a chiral unit with a rigid bend body. Elongation of the peptide chain significantly increased the CD intensity per phenylene chromophore; however, the CD intensity decreased at high temperatures. This cooperative behavior strongly suggested the formation of a secondary structure. 1H NMR analysis revealed a right-handed helical structure in dimethyl sulfoxide. The theoretical calculations were consistent with these results.

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INTRODUCTION Biological macromolecules, e.g., proteins and nucleic acids form well-defined three-dimensional structures, which provide unique, sophisticated, and precise functions such as molecular recognition, information storage, and catalysis. Biological macromolecules with chiral centers provide asymmetric environments for accurate molecular recognition and enantioselectivity. Many researchers have reported various kinds of oligomers and polymers forming well-defined three-dimensional structures, which are called foldamers. Each molecule exhibits a compact folded structure formed via intramolecular π−π interactions or stacking, steric hindrance, hydrogen bonds, electrostatic interactions, and so on.1−23 On the other hand, chiral polymers with an artificial stiff backbone have been synthesized, e.g., poly(isocyanide)s, poly(isocyanate)s, and poly(acetylene)s.24−30 These polymers form stable helices because of the restricted torsion angle between neighboring repeat units. In the absence of any sterically predominant factor and if the energy barrier is sufficiently low to allow helix inversion, right and left helical senses are equal population. Poly(isocyanate)s made from achiral monomers show this behavior. Addition of very small amounts of a chiral compound to racemic helices or retention of the delicate balance between two optical isomers provides one-handedness keeping the original helicity. In both dynamic and static helical polymers, each monomer unit prefers the helical sense, but the difference between the energy levels for two helical senses should be minimal, unless a predominant factor is present, which could result in high sensitivity of the helical sense to external factors. The cooperative motion and folding, as observed in helix− coil transition, are essential to biological functions involving supramolecular or allosteric interactions. The cooperativity is © XXXX American Chemical Society

provided by repeated and sequential changes with a relatively low-energy barrier for each deviation. Proteins are among the most refined and perfect molecules for this purpose. Enantioselective reactions are facilitated by the chiral active sites in chiral proteins, which are constructed predominantly by L-α-amino acids with inherent chirality. In a typical protein, two neighboring planes are connected by one Cα carbon; thus, the torsion angles (ϕ, ψ) are allowed for each amino acid residue, as shown in the Ramachandran plot.31,32 A possible region of (ϕ, ψ) is limited by the chirality of the Cα carbon and the type of side chain. The secondary structure of a protein is determined by the primary structure in the amino acid sequence. A newly biosynthesized peptide chain automatically forms a roughly ordered intrinsic three-dimensional structure. The preorganized secondary structure is stabilized by intramolecular hydrogen bonds, electrostatic interactions, and hydrophobic interactions to complete the final folding structure. A recent study demonstrated that the handedness of the asymmetric environment derived from an artificial helical scaffold dominates the enantioselectivity of the embedded catalytic center.33 Natural proteins afford extremely large numbers of asymmetric environments in enzymes; however, they rely on a limited repertoire of amino acids. To construct a newly ordered achiral environment, the limited repertoire is obstacle but still tailor-made designs of building blocks are preparatory. And incorporation of a functional group at the side chain or main chain of an established macromolecular structure requires a lot of effort and time. If the repertoire is expanded, infinite combination is expected. Received: April 6, 2017

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DOI: 10.1021/acs.macromol.7b00718 Macromolecules XXXX, XXX, XXX−XXX

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of the monomer H-Leu-NHC6H4COOH using water-soluble carbodiimide (WSCD), followed by capping of the free amino and carboxyl groups at the N- and C-terminals. 1H NMR spectra of 1−5 show peaks due to four kinds of amide protons, as shown in Figure 2 in distinct colors. The blue-colored

In this paper, we present a new strategy for constructing chiral helices, expanded poly(α-amino acid)s, as mentioned in the previous paper.34 Each natural L-α-amino acid contained in proteins shows individual tendency to form a secondary structure. For example, leucine is well-known for exhibiting high helix-forming tendency, while poly(L-leucine)s form a stable right-handed α-helix, as found in the leucine zipper.32 A rigid spacer, p-phenylene in this study, was inserted between two leucine residues of poly(L-leucine)s via amide linkage, as shown in Figure 1. The original amide plane and the rigid

Figure 1. Schematic drawing of (a) expanded L-leucine containing pphenylene moieties and (b) expanded oligo- and polyl(L-leucine)s (R = t-Bu, 1−5; R = CH3, models for calculations, 1′−5′).

spacer formed a new rigid group called “expanded amide”. To extract the essence of the torsional or steric effects of the side chain, inter- and intramolecular hydrogen bonds were excluded. The obtained expanded poly-L-leucine was dissolved in a polar solvent, i.e., dimethyl sulfoxide (DMSO), which cleaves the hydrogen bonds by strong solvation. Circular dichroism (CD) spectra strongly suggested the formation of a helical structure in DMSO, as was determined by 1H NMR analysis. Theoretical calculations using the density functional theory (DFT) provided the validity of the proposed structure and satisfactorily simulated the observed spectra. In recent years, some combinations of aromatic oligoamide frames and α-amino acids have been reported for the development of linear arrays for protein surface recognition.35,36

Figure 2. 1H NMR spectra of (a) 1, (b) 2, (c) 3, (d) 4, and (e) 5 in DMSO-d6 at 30 °C.

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protons are found at 10.31 and 10.25 ppm. The latter is the Cterminal anilide NH proton. The former represents the protons at the intermediate positions. The intensity ratio of these peaks is consistent with the degree of polymerization (n). The increase in integral intensity of the monomer unit relative to the terminal t-Bu and NH groups clearly shows the steady elongation of the peptide chain. The unchanged peak positions and similarity of these spectra indicate that the electronic and magnetic environments are not dependent on the length of the peptide chain and suggest that each monomer unit exerts minimal effect on the neighboring units. Characterization of Polymer 5 by SEC. Size-exclusion chromatography (SEC) was used to determine the degree of polymerization of 5. As a conventional and convenient method, SEC is used widely to estimate the mean value and distribution of molecular weight of a polymer. Generally, polystyrene (PS) is used as a standard polymer to obtain the calibration curve and to evaluate the molecular weight from the elution volume.

RESULTS AND DISCUSSION Synthesis. A series of expanded oligo(L-leucine)s were synthesized by stepwise elongation and fragment coupling using a conventional method for liquid-phase peptide synthesis, as shown in Scheme S1. Because the amino group in aminobenzoic acid has relatively low basicity, which leads to low nucleophilic reactivity, the peptide linkage between Leu and −NHC6H4CO− was formed in the first step and the −LeuNHC6H4CO− unit was used as a building block. The Nterminal t-BuCONHC6H4CO− group was introduced in the final step. For octamer 4, a similar synthetic procedure as that for 3 involved a difficult separation step because of the low solubility of the deprotected octamer in acidic aqueous solution. The fragment condensation shown in Scheme S1 and subsequent repeated reprecipitation from DMF/THF gave a satisfactory product. Polymer 5 was prepared by condensation B

DOI: 10.1021/acs.macromol.7b00718 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules SEC analysis using N,N-dimethylformaldehyde (DMF) with 0.1 M LiBr and commercially available standard PS did not give reasonable values for oligomers 1−4, which were fully characterized by 1H NMR and ESI-MS. The elution curves are shown in Figure 3a. For oligomers 1−4, the logarithmic

Figure 4. UV (a) and CD (b) spectra of 1 (black), 2 (red), 3 (blue) 4 (green), and 5 (purple) in DMSO at 30 °C. A plot of the peak top against the degree of polymerization; n is shown in the inset of (b).

The split pattern (+ to − ) of the CD spectra at about 270 nm is assigned to exciton coupling between two chromophores with clockwise spin spiral arrangement based on the exciton chirality method.37 This Cotton effect corresponds to the absorption maximum in Figure 4a, and the negative intensity at about 260 nm depends to a smaller extent on the length of the peptide chain. These results suggest that the Cotton effect is caused by the restricted local conformation of two phenylene−amide planes adjacent to one chiral Cα atom. As illustrated in Figure S1, two polarized transition moments along the long axis of each 4-aminobenzoyl chromophore are located in the clockwise twist. In contrast, the maxima at 290 nm are enhanced by the elongation of the peptide chain. The dependence of the maxima on the peptide chain elongation is shown in the inset of Figure 4b. The peak top is shifted to longer wavelength, which agrees with the redshift of the shoulder peak in the absorption spectra (Figure 4a). This kind of positive Cotton effect without typical exciton splitting predominantly arises from electron movement along a right-handed (P) helix, as mentioned in previous papers.34,38,39 Moreover, the enhancement of the CD signal per phenylene moiety indicates the formation of a well-defined helical structure.34 In the case of N-alkylated poly(p-benzamide)s, which have a similar 4-aminobenzoyl chromophore and form a P-helix, a similar positive Cotton effect at the longest wavelength absorption depending on molecular weight was reported.13 The sign of the Cotton effect depends on the direction of the dipole moments of the chromophores and does not directly indicate P or M for the whole helix. A positive-tonegative split or positive Cotton effect indicates P-helix in our case, but in the case of an usual peptide or dehydroamino acid,40 it should be M-helix. To confirm the handedness estimated from CD spectra, theoretical calculations are effective41 as described in the section DFT Calculations. L-leucine.

Figure 3. (a) SEC elution curves of 1−5. (b) A logarithmic plot of the molecular weight of 1−4 and estimated mean molecular weight of 5 against the elution volume.

plot of true molecular weight against elution volume was found to be linear, as shown in Figure 3b. The calculated mean molecular weight based on this linear correlation was 4.7 × 103 (n ≃ 19). Analysis of the distribution curve gave a weightaverage molecular weight (Mw) and number-average molecular weight (Mn) of 6.9 × 103 and 3.4 × 103 (PS standards), respectively. The Mw/Mn ratio was 2.0, which is an ideal value for typical polycondensation in high extent of reaction or high conversion to polymer. The degree of polymerization (n) is consistent with the estimated value from the 1H NMR spectra. UV and CD Spectra. Absorption (UV) and CD spectra of 1−5 in DMSO are shown in Figure 4. The vertical axis (ε or Δε) is normalized by the concentration of the phenylene unit, where the conjugated moiety of phenylene and amide planes make up the principal chromophore. The π−π* transition of the aromatic group and intramolecular charge transition (CT) bands were observed at 250−350 nm. The shapes of these spectra resemble each other, which suggests that each monomer unit is independent and that a significant electronic interaction between interunit is little in the ground state, as revealed in the 1H NMR spectra. The slight shift of the absorption maxima to the lower energy side by the elongation of the peptide chain was probably caused by the weak interunit interaction and the formation of the secondary structure. A theoretical approach to reveal the origin of this red-shift is shown in another section concerning DFT calculations. The spectra in Figure 4b show the induced CD corresponding to the absorption band of the achiral −CONHC6H4CONH− moiety arising from the chiral Cα of C

DOI: 10.1021/acs.macromol.7b00718 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules Temperature Dependence of CD Spectra. To check the formation and flexibility of the helix, the temperature dependence of the CD spectra was examined. The CD and absorption spectra of 5 at 30−80 °C are shown in Figure 5. The

Figure 6. Temperature dependence of the peak top in (a) UV and (b) CD spectra of 3−5.

Cotton effect at longer wavelengths is caused by the formation of a well-defined secondary structure of the peptide. Molecular Structure in Solution. The molecular structure of polymer 5 was simulated by using structural information from 1H NMR spectra of 3 and molecular dynamics calculations. The procedure is essentially similar to a reported method.34 The NOESY spectrum of 3 in DMSO-d6 is shown in Figure S3a. Observed NOEs are shown as red circled lines with the assignment, which are also shown as red arrows in Figure S3b. Based on the observed NOEs related to the leucine residue, 15 distance restraints were set. Dihedral angles obtained from 3J coupling were used for the restraints. Molecular dynamics using the simulated annealing method (SA) method gave 50 structures satisfying the restraint conditions. Using the lowest structure, 19-mer was constructed. Energy minimization of the structure resulted in a right-handed helix, as shown in Figure 7a,b. The length and width are 102 and 13 Å, respectively. The helix contains a little over 3 residues per turn, approximately 10/3 or 31 helix, which is between the 310 and α-helix of natural peptides. The pitch (about 19 Å) is significantly longer than that of α-helix. Polymer 5 exists as a helix in DMSO, which breaks the intra- and intermolecular hydrogen bonds, thereby preventing uncontrolled aggregation. Such an environment permits the restrained rigid body of the expanded L-leucine residue to determine the whole secondary structure. The steady conformation of the expanded L-leucine residue is supported by the stable negative Cotton effect in the CD spectra at high temperatures. DFT Calculations. DFT calculation was performed to reveal the origin of the long-wavelength shift in the absorption spectra by the elongation of peptide chain. First, geometrical optimizations were performed. The polarity of the solvent plays a very important role in the determination of the solution structure. The optimization of 1′ (Figure 1) in vacuum or chloroform resulted in an unsuitable structure with an unusually short intramolecular NH···OC contact. The

Figure 5. Temperature dependence of (a) UV and (b) CD spectra of 5 in DMSO.

intensity of the absorption maximum was slightly decreased at higher temperatures. In particular, a decrease in the shoulder peak at longer wavelengths was observed. The intensity of the CD spectra decreased significantly at higher temperature, suggesting the flexibility of the helix.13 Interestingly, the decrease in Cotton effect was limited to longer wavelengths. The negative Cotton effect at 260 nm was unchanged. Based on the discussion in the previous section, these results suggest that the local conformation around the Cα atom is fixed, but the overall conformation is flexible at higher temperatures. Thus, the orientation of two neighboring phenylene planes is determined by the side chain of the leucine residue to form a rigid bent unit, which is connected to the adjacent unit by a rotatable phenylene−amide bond. Although the single bond is freely rotatable, the two amide groups connected to both sides of the phenylene moiety should be oriented opposite to each other and retain coplanarity with the phenylene plane at the energy minimum. The total helicity is probably determined by thermodynamic stability with statistical fluctuation. Plots of ε and Δε of 5 against temperature are shown in Figure 6 and compared with those for tetramer 3 and octamer 4. For the absorption coefficient, the slope of 5 is negative and small, which resembles that of 3 and 4. In the case of Δε, the slope of 5 shows a sharper downward trend than that of 3 and 4. The classic Zimm−Bragg model shows that the statistical weight is approximately proportional to temperature near the transition point.32,42 Though the linearity should be applicable to a limited range of temperature, extrapolation of these lines shows the point of intersection at Δε = 7−8 and at 160−170 °C, as depicted in Figure S2. The CD spectra of 3−5 are expected to approach that of monomer 1. These results strongly suggest the D

DOI: 10.1021/acs.macromol.7b00718 Macromolecules XXXX, XXX, XXX−XXX

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normalized intensities per phenylene moiety (i.e., the values divided by 2 for 1′, 3 for 2′, and 5 for 3′) were almost the same (Figures S4 and S5, Figure 8). These results are consistent with the observation that a longer wavelength shift occurs with an increase in the number of monomer units. The second and third strongest absorption bands are essentially of the same energy at 268 and 259 nm. On the other hand, the normalized CD intensity obviously increases by the elongation of the peptide chain, as 1′ < 2′ < 3′, which is also consistent with the observations. The predominant transitions and molecular orbitals (MOs) of 1′−3′ are illustrated in Figure S5. The lowest energy absorption is essentially due to the transition from the higher filled orbital to the LUMO. The LUMO is extended over a wide range of the molecule, as in a conjugated system. Such an orbital should be stabilized by the elongation of the molecule. The energy level of the LUMO was lowered in the order 3′ (−0.048 32 au) < 2′ (−0.047 32 au) < 1′ (−0.045 44 au), although the HOMO energy was less affected or was identical, 3′ (−0.230 05 au) ≤ 2′ (−0.229 94 au) ≤ 1′ (−0.229 82 au). On the other hand, the MOs for the other absorption bands at 268 and 259 nm were localized within one residue. The normalized intensity of the simulated CD spectra increased, especially at longer wavelengths, in the order 3′ > 2′ > 1′. Because the transition occurs parallel to the helical axis, it is enhanced by the elongation of the molecule along the axis. In a real solution, the CD signal fluctuates because of the thermal motion of the molecule. The lowest-energy inter-residue transition is usually influenced by the molecular dynamics, although this was not taken into account in the simulation of the CD spectra.

Figure 7. Proposed solution structures of 5′ from the 1H NMR analysis of 3 (a, side; b, top) and from the optimized structure of 3′ using DFT calculations (c, side; d, top).

optimization in DMSO afforded a reasonable structure, and time-dependent (TD) DFT calculations provided the validity of the structure. The initial model of 2′ was constructed from the optimized structure of 1′ and optimized. The structure of 3′ was determined in a similar manner using the optimized 2′. Unfortunately, the geometrical optimization of 4′ could not meet convergence despite many trials. A proposed structure of 19-mer 5′ based on the optimized structure of 3′ is shown in Figure 7c,d. The whole structure is a right-handed 13/4 (≈ 31) helix, similar to the extended structure revealed by the NMR spectrum in Figure 7a. These results, together with the simulated CD spectra, strongly indicate that polymer 5 forms an approximate 31 helix in DMSO. The simulated UV and CD spectra of 3′ were quite similar to the observed spectra, although the intensity was overestimated (Figure 8). The calculated lowest-energy transition of 1′−3′ was at 272.92, 275.79, and 277.43 nm with an oscillator strength (f) of 0.9341, 1.4181, and 2.5199, respectively. The

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CONCLUSIONS Mono-, di-, tetra-, octa-, and polymers (1−5) of expanded Lleucine were synthesized by stepwise elongation or polycondensation using the typical liquid-phase peptide synthesis. Products 1−5 were characterized by 1H NMR, ESI-MS, SEC, UV, and CD spectra. 1H NMR spectra showed every slight electronic interunit interactions, but UV spectra suggested the presence of slight interactions at longer wavelengths. Enhancement of the intensity of Cotton effects with the red-shift of the absorption maxima by elongation of the peptide chain indicated the growth of a well-defined secondary structure. The temperature dependence of the CD spectrum of 5 shows a steady intensity at shorter wavelengths and decreased intensity at the longer wavelengths. These results suggest that the former is due to intraunit transition within the rigid monomer unit and the latter is due to the transition between monomer units with a variable orientation, as is strongly supported by theoretical calculations. Two-dimensional 1H NMR analysis and the spectral simulation of the optimized structure using DFT calculations revealed the presence of a right-handed helix. From these results, the following general conclusions can be drawn. In the expanded poly(α-amino acid)s, the local conformation of each monomer unit is determined by the type of the constituent α-amino acid, that is, steric and/or electrostatic effects of the side chain. In other words, restriction of the torsion angles (ϕ, ψ) is essential in determining the secondary structure. In this study, selection of L-leucine, which prefers an α-helix, resulted in successful construction of a helical structure. As a natural α-amino acid shows a tendency to form a secondary structure, each expanded α-amino acid should have an individual tendency arising from the original chemical structure. Appropriate selection and combination of expanded

Figure 8. Simulated UV (a) and CD (b) spectra of 3′ in DMSO. The vertical axis is divided by the number of phenylene moieties, 5. E

DOI: 10.1021/acs.macromol.7b00718 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules α-amino acids are important in the construction of an expanded protein with a unique tertiary structure. As shown in a previous paper,34 metal complexes can be introduced to a rigid group. The replacement of the phenylene moiety with various functional groups and/or L-leucine residue with various αamino acids, including non-natural amino acids, will give access to numerous types of expanded proteins. Our strategy described in this paper is expected to aid in the development of new effective procedures for the synthesis of expanded proteins.

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The solution was stirred for 24 h at room temperature. After removal of solvents under reduced pressure, the residue was dissolved in water. The solution was washed with ethyl acetate, and the aqueous layer was acidified with citric acid to give an oily product which was extracted with ethyl acetate. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was reprecipitated from hot ethyl acetate, collected with filtration, and dried in vacuo. Yield 2.0 g (73%). 1H NMR (DMSO-d6): δ 0.87 (d, 3H, J = 1.6 Hz, Leu-δ-(CH3)2), 0.90 (d, 3H, J = 1.6 Hz, Leu-δ-(CH3)2), 1.37 (s, 9H, Boc), 1.42−1.64 (m, 3H, Leu-β-CH2, γ-CH), 4.13 (br, 1H, Leu-αCH), 7.05 (d, 1H, J = 7.8 Hz, Leu-NH), 7.70 (d, J = 8.9 Hz, 2H, Ar− H), 7.88 (d, J = 8.9 Hz, 2H, Ar−H), 10.22 (s, 1H, Boc-Leu-NHC6H4), 12.63 (br, 1H, COOH). ESI-MS Calcd for C18H26N2O5 ([M + Na]+): 373.2; found: 373.1, ([M − H]−): 349.1; found 349.0. Boc-Leu-NHC6H4CONH-t-Bu. Compound 8 (200 mg, 0.57 mmol) and HOBt (92 mg, 0.68 mmol) were dissolved in DMF (4 mL). To the solution was added tert-butylamine (0.1 mL, 0.94 mmol) and WSCD (0.12 mL, 0.66 mmol) in an ice bath. After stirring for 1.5 h in an ice bath and overnight at room temperature, the solution was concentrated to dryness under reduced pressure. The residue was dissolved in ethyl acetate. The solution was washed with saturated NaCl(aq), 4% NaHCO3(aq), saturated NaCl(aq), 10% citric acid(aq), and saturated NaCl(aq), successively, and dried over Na2SO4. After removal of solvents under reduced pressure, the residue was reprecipitated from ethyl acetate and n-hexane and dried in vacuo. Yield 170 mg (74%). 1H NMR (DMSO-d6): δ 0.87 (d, 3H, J = 1.4 Hz, Leu-δ-(CH3)2), 0.90 (d, 3H, J = 2.2 Hz, Leu-δ-(CH3)2), 1.36 (s, 9H, N-t-Bu), 1.37 (s, 9H, Boc), 1.60 (m, 3H, Leu-β-CH2, γ-CH), 4.12 (br, 1H, Leu-α-CH), 7.00 (d, 1H, J = 8.1 Hz, Leu-NH), 7.56 (s, 1H, NH-tBu), 7.62 (d, J = 8.8 Hz, 2H, Ar−H), 7.75 (d, J = 8.8 Hz, 2H, Ar−H), 10.07 (s, 1H, Boc-Leu-NHC6H4). HCl·H-Leu-NHC6H4CONH-t-Bu. This compound was synthesized by a similar method as described for HCl·H-Leu-NHC6H4COOEt. Yield 128 mg (89%).1H NMR (DMSO-d6): δ 0.93 (m, 6H, Leu-δ-(CH3)2), 1.36 (s, 9H, N-t-Bu), 1.68 (m, 3H, Leu-β-CH2, γ-CH), 4.00 (br, 1H, Leu-α-CH), 7.61 (s, 1H, NH-t-Bu), 7.66 (d, J = 8.6 Hz, 2H, Ar−H), 7.80 (d, J = 8.6 Hz, 2H, Ar−H), 8.33 (br, 3H, Leu-NH3Cl), 10.88 (br, 1H, Leu-NHC6H4). t-BuCONHC 6 H 4 CO-Leu-NHC 6 H 4 CONH-t-Bu (1). HCl·H-LeuNHC6H4CONH-t-Bu (46 mg, 0.14 mmol), 6 (30 mg, 0.14 mmol), and HOBt (22 mg, 0.14 mmol) were dissolved in DMF (2 mL). To the solution were added 2 drops of triethylamine in an ice bath and 3 drops of WSCD at −15 °C. After stirring for 1.5 h at the temperature and overnight at room temperature, the solvents were removed under reduced pressure. The residue was dissolved in ethyl acetate. The solution was washed with saturated NaCl(aq), 4% NaHCO3(aq), saturated NaCl(aq), 2% HCl(aq), and saturated NaCl(aq) successively and dried over Na2SO4. After removal of solvents under reduced pressure, the residue was reprecipitated from ethyl acetate and nhexane. The powder was collected with filtration and dried in vacuo. Yield 30 mg (43%). 1H NMR (DMSO-d6): δ 0.91 (d, 3H, J = 6.8 Hz, Leu-δ-(CH3)2), 0.94 (d, 3H, J = 6.5 Hz, Leu-δ-(CH3)2), 1.23 (s, 9H, tBuCO), 1.36 (s, 9H, N-t-Bu), 1.56−1.75 (m, 3H, Leu-β-CH2, γ-CH2), 4.64 (br, 1H, Leu-α-CH), 7.56 (s, 1H, NH-t-Bu), 7.65 (d, J = 8.9 Hz, 2H, Ar−H), 7.73 (d, J = 8.6 Hz, 2H, Ar−H), 7.76 (d, J = 8.9 Hz, 2H, Ar−H), 7.87 (d, J = 8.6 Hz, 2H, Ar−H), 8.43 (d, 1H, J = 8.4 Hz, LeuNH), 9.37 (s, 1H, t-BuCONHC6H4), 10.25 (s, 1H, Leu-NHC6H4). ESI-MS Calcd for C29H40N4O4 ([M + Na]+): 531.3; found 531.3, ([M − H]−): 507.3; found 507.3. Boc-(Leu-NHC6H4CO)2OEt (9). This compound was synthesized from 8 (1.9 g, 5.4 mmol) and HCl·H-Leu-NHC6H4COOEt (1.7 g, 5.4 mmol) by a similar method as described for 7. Yield 1.9 g (57%). 1H NMR (DMSO-d6): δ 0.92 (m, 12H, Leu-δ-(CH3)2), 1.30 (t, 3H, J = 7.2 Hz, CH3), 1.38 (s, 9H, Boc), 1.42−1.77 (m, 6H, Leu-β-CH2, γCH), 4.13 (br, 1H, Leu-α-CH), 4.27 (q, 2H, J = 7.2 Hz, CH2), 4.64 (br, 1H, (C6H4CO−)Leu-α-H), 7.02 (d, 1H, J = 8.0 Hz, (Boc-)LeuNH), 7.67 (d, J = 8.4 Hz, 2H, Ar−H), 7.76 (d, J = 8.8 Hz, 2H, Ar−H), 7.87 (d, J = 8.4 Hz, 2H, Ar−H), 7.90 (d, J = 8.8 Hz, 2H, Ar−H), 8.46 (d, 1H, J = 8.0 Hz, (C6H4CO−)Leu-NH), 10.13 (s, 1H, Boc-LeuNHC6H4), 10.41 (s, 1H, C6H4CO-Leu-NHC6H4).

EXPERIMENTAL SECTION

Materials. All solvents except ethanol were dried over calcium hydride and distilled under an argon atmosphere prior to use. N-(tButoxycarbonyl)-L-leucine·monohydrate (Boc-Leu-OH·H2O), 1hydroxybenztriazole (HOBt), and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (WSCD) were purchased from Protein Research Foundation, Osaka. Other reagents were purchased from Nacalai Tesque Inc. (Kyoto, Japan) or Tokyo Chemical Industry. t-BuCONHC6H4COOH (6). p-Aminobenzoic acid (1.7 g, 12 mmol) was dissolved in a mixture of CH3CN (20 mL) and THF (2 mL). To the solution were added triethylamine (3.4 mL, 24 mmol) and pivaloyl chloride (3 mL, 24 mmol) in an ice bath. After stirring for 48 h, the solvents were removed under reduced pressure. The residue was dissolved in ethanol (20 mL). To the solution was added 1 M NaOH(aq) (14 mL) in an ice bath. After stirring for overnight, the solvents were removed under reduced pressure. The residue was dissolved in ethyl acetate, washed with saturated NaCl(aq), 2% HCl(aq), and saturated NaCl(aq) successively, and dried over Na2SO4. After removal of solvents, the residue was recrystallized from ethanol. The product was collected with filtration, washed with small amount of ethanol, and dried in vacuo. Yield 0.81 g (30%). 1H NMR (DMSO-d6): δ 1.23 (s, 9H, t-Bu), 7.78 (d, 2H, J = 8.5 Hz, Ar− H), 7.86 (d, 2H, J = 8.5 Hz, Ar−H), 9.43 (s, 1H, NH), 12.57 (br, 1H, COOH). Boc-Leu-NHC6H4COOEt (7). Ethyl p-aminobenzoate (3.7 g, 22 mmol) and Boc-Leu-OH·H2O (5.5 g, 22 mmol) were dissolved in CH2Cl2 (15 mL). To the solution was added N,N′-dicyclohexylcarbodiimide (DCC) (5.0 g, 24 mmol) cooling in an ice bath. The solution was stirred in an ice bath for 2.5 h and at room temperature overnight. After filtering out the precipitate, the filtrate was concentrated to dryness under reduced pressure. The residual oil was dissolved in ethyl acetate. The solution was washed successively with saturated NaCl(aq), 4% NaHCO3(aq), saturated NaCl(aq), 10% citric acid(aq), and saturated NaCl(aq), and dried over Na2SO4. After removal of solvents under reduced pressure, the residue was recrystallized from diethyl ether and n-hexane. The white powder was collected with filtration and dried in vacuo. Yield 5.4 g (65%). 1H NMR (DMSO-d6): δ 0.88 (d, 3H, J = 2.4 Hz, Leu-δ-(CH3)2), 0.89 (d, 3H, J = 2.8 Hz, Leu-δ-(CH3)2), 1.30 (t, 3H, J = 7.2 Hz, CH3), 1.37 (s, 9H, Boc), 1.53−1.65 (m, 3H, Leu-β-CH2, γ-CH), 4.13 (br, 1H, Leu-αCH), 4.27 (q, 2H, J = 7.2 Hz, CH2), 7.05 (d, 1H, J = 8.8 Hz, Leu-NH), 7.73 (d, J = 8.8 Hz, 2H, Ar−H), 7.90 (d, J = 8.8 Hz, 2H, Ar−H), 10.25 (s, 1H, Leu-NHC6H4). Anal. Calcd for C20H30N2O5: C, 63.47; H, 7.99; N, 7.40. Found: C, 63.44; H, 7.98; N, 7.45. HCl·H-Leu-NHC6H4COOEt. To a solution of 7 (2.4 g, 6.3 mmol) in ethyl acetate (5 mL) was added saturated HCl−ethyl acetate solution (106 mL) in an ice bath. After stirring overnight at room temperature, the solution was concentrated to dryness under reduced pressure. The residue was washed with diethyl ether and dried in vacuo. Yield 2.0 g (86%). 1H NMR (DMSO-d6): δ 0.92 (d, 3H, J = 2.8 Hz, Leu-δ(CH3)2), 0.94 (d, 3H, J = 3.6 Hz, Leu-δ-(CH3)2), 1.31 (t, 3H, J = 7.2 Hz, CH3), 1.68 (m, 3H, Leu-γ-CH, ββ-CH2), 4.06 (m, 1H, Leu-αCH), 4.28 (q, 2H, J = 7.2 Hz, CH2), 7.79 (d, J = 7.8 Hz, 2H, Ar−H), 7.95 (d, J = 7.8 Hz, 2H, Ar−H), 8.40 (br, 3H, Leu-NH3Cl), 11.20 (br, 1H, Leu-NHC6H4). Boc-Leu-NHC6H4COOH (8). To a solution of 7 (3.1 g, 8.3 mmol) in ethanol (12 mL) was added 1 M NaOH(aq) (9.2 mL) in an ice bath. F

DOI: 10.1021/acs.macromol.7b00718 Macromolecules XXXX, XXX, XXX−XXX

Article

Macromolecules Boc-(Leu-NHC6H4CO)2OH (10). This compound was synthesized from 9 (1.9 g, 3.1 mmol) by a similar method as described for 8. Yield 0.81 g (77%). 1H NMR (DMSO-d6): δ 0.92 (m, 12H, Leu-δ-(CH3)2), 1.38 (s, 9H, Boc), 1.42−1.77 (m, 6H, Leu-β-CH2, γ-CH), 4.13 (br, 1H, Leu-α-CH), 4.65 (br, 1H, (C6H4CO−)Leu-α-H), 7.02 (d, 1H, J = 8.8 Hz, (Boc-)Leu-NH), 7.67 (d, J = 8.8 Hz, 2H, Ar−H), 7.73 (d, J = 8.8 Hz, 2H, Ar−H), 7.87 (d, J = 8.8 Hz, 4H, Ar−H), 8.46 (d, 1H, J = 7.2 Hz, (C6H4CO−)Leu-NH), 10.13 (s, 1H, Boc-Leu-NHC6H4), 10.38 (s, 1H, C6H4CO-Leu-NHC6H4), 12.63 (br, 1H, COOH). Boc-(Leu-NHC6H4CO)2NH-t-Bu (11). This compound was synthesized from 10 (1.26 g, 2.16 mmol) by a similar method as described for Boc-Leu-NHC6H4CONH-t-Bu. Yield 1.0 g (73%). 1H NMR (DMSO-d6): δ 0.91 (m, 12H, Leu-δ-(CH3)2), 1.36 (s, 9H, N-t-Bu), 1.37 (s, 9H, Boc), 1.42−1.77 (m, 6H, Leu-β-CH2, γ-CH), 4.12 (br, 1H, (Boc-)Leu-α-CH), 4.64 (br, 1H, (C6H4CO−)Leu-α-CH), 7.02 (d, 1H, J = 8.4 Hz, (Boc-)Leu-NH), 7.56 (s, 1H, NH-t-Bu), 7.65 (d, J = 8.9 Hz, 2H, Ar−H), 7.67 (d, J = 8.9 Hz, 2H, Ar−H), 7.76 (d, J = 8.9 Hz, 2H, Ar−H), 7.88 (d, J = 8.9 Hz, 2H, Ar−H), 8.43 (d, 1H, J = 7.6 Hz, (C6H4CO−)Leu-NH), 10.13 (s, 1H, Boc-Leu-NHC6H4), 10.25 (s, 1H, -Leu-NHC6H4). HCl·H-(Leu-NHC6H4CO)2NH-t-Bu (12). Compound 11 (0.55 g, 0.86 mmol) was dissolved in CH2Cl2 (1.9 mL). To the solution was added TFA (1.9 mL, 26 mmol) in an ice bath. After stirring overnight at room temperature under an argon atmosphere, the solvents were removed under reduced pressure. To the residual oil was added saturated HCl−ethyl acetate solution in an ice bath. Then the solvents were evaporated. This operation was repeated three times. The obtained powder was washed with ethyl acetate. Yield 0.43 g (89%). 1 H NMR (DMSO-d6): δ 0.93 (m, 12H, Leu-δ-(CH3)2), 1.36 (s, 9H, N-t-Bu), 1.68−1.77 (m, 6H, Leu-β-CH2, γ-CH), 3.95 (br, 1H, (HCl· H−)Leu-α-CH), 4.66 (br, 1H, (C6H4CO−)Leu-α-CH), 7.56 (s, 1H, NH-t-Bu), 7.65 (d, J = 8.9 Hz, 2H, Ar−H), 7.68 (d, J = 8.9 Hz, 2H, Ar−H), 7.76 (d, J = 8.9 Hz, 2H, Ar−H), 7.94 (d, J = 8.9 Hz, 2H, Ar− H), 8.24 (br, 3H, Leu-NH3Cl), 8.51 (d, 1H, J = 7.6 Hz, Leu-NH), 10.29 (s, 1H, H-Leu-NHC6H4), 10.71 (s, 1H, -Leu-NHC6H4). t-BuCONHC6H4CO-(Leu-NHC6H4CO)2NH-t-Bu (2). This compound was synthesized from 12 by a similar method as described for 1. Yield 122 mg (75%) 1H NMR (DMSO-d6): δ 0.94 (m, 12H, Leu-δ-(CH3)2), 1.23 (s, 9H, t-BuCO), 1.36 (s, 9H, N-t-Bu), 1.56−1.80 (m, 6H, Leu-βCH2, γ-CH), 4.65 (br, 2H, Leu-α-CH), 7.56 (s, 1H, NH-t-Bu), 7.65 (d, J = 8.6 Hz, 2H, Ar−H), 7.70 (d, J = 8.9 Hz, 2H, Ar−H), 7.74 (d, J = 8.9 Hz, 2H, Ar−H), 7.76 (d, J = 8.6 Hz, 2H, Ar−H), 7.87 (d, J = 8.9 Hz, 2H, Ar−H), 7.88 (d, J = 8.9 Hz, 2H, Ar−H), 8.44 (d, 2H, J = 7.4 Hz, Leu-NH), 9.37 (s, 1H, t-BuCONHC6H4), 10.25 (s, 1H, tBuCONHC6H4CO-Leu-NHC6H4), 10.31 (s, 1H, Leu-NHC6H4). ESIMS Calcd for C42H56N6O6 ([M + Na]+): 763.4; found 763.5, ([M − H]−): 739.4; found 739.3. Boc-(Leu-NHC6H4CO)4NH-t-Bu. This compound was synthesized from 10 and 12 by a similar method as described for 9. Yield 205 mg (57%). 1H NMR (DMSO-d6): δ 0.92 (m, 24H, Leu-δ-(CH3)2), 1.36 (s, 9H, N-t-Bu), 1.37 (s, 9H, Boc), 1.42−1.75 (m, 12H, Leu-β-CH2, γCH), 4.13 (br, 1H, (Boc-)Leu-α-CH), 4.64 (br, 3H, Leu-α-CH), 7.03 (d, 1H, J = 7.6 Hz, (Boc-)Leu-NH), 7.56 (s, 1H, NH-t-Bu), 7.65 (d, J = 8.6 Hz, 2H, Ar−H), 7.67 (d, J = 8.6 Hz, 2H, Ar−H), 7.70 (d, J = 8.6 Hz, 4H, Ar−H), 7.75 (d, J = 8.6 Hz, 2H, Ar−H), 7.88 (d, J = 8.6 Hz, 6H, Ar−H), 8.45 (m, 3H, Leu-NH), 10.13 (s, 1H, Boc-Leu-NHC6H4), 10.25, 10.30 (s, 3H, CO-Leu-NH-C6H4). HCl·H-(Leu-NHC6H 4CO)4 NH-t-Bu (13). This compound was synthesized from Boc-(Leu-NHC6H4CO)4NH-t-Bu by a similar method as described for 12. Yield 132 mg (88%). 1H NMR (DMSO-d6): δ 0.93 (m, 24H, Leu-δ-(CH3)2), 1.36 (s, 9H, N-t-Bu), 1.58−1.80 (m, 12H, Leu-β-CH2, γ-CH), 3.97 (br, 1H, (HCl·H−)Leuα-CH), 4.65 (br, 3H, Leu-α-CH), 7.56 (s, 1H, NH-t-Bu), 7.65 (d, J = 8.6 Hz, 2H, Ar−H), 7.70 (d, J = 8.6 Hz, 6H, Ar−H), 7.76 (d, J = 8.6 Hz, 2H, Ar−H), 7.89 (d, J = 8.6 Hz, 4H, Ar−H), 7.95 (d, J = 8.6 Hz, 2H, Ar−H), 8.27 (br, 3H, Leu-NH3Cl), 8.49 (m, 3H, Leu-NH), 10.26, 10.33, 10.36 (s, 3H, CO-Leu-NHC6H4), 10.77 (br, 1H, H-LeuNHC6H4). t-BuCONHC6H4CO-(Leu-NHC6H4CO)4NH-t-Bu (3). This compound was synthesized from 13 by a similar method as described for 1. Yield

35 mg (50%). 1H NMR (DMSO-d6): δ 0.93 (m, 24H, Leu-δ-(CH3)2), 1.23 (s, 9H, t-BuCO), 1.36 (s, 9H, N-t-Bu), 1.56−1.77, (m, 12H, Leuβ-CH2, γ-CH), 4.65 (br, 4H, Leu-α-CH), 7.56 (s, 1H, NH-t-Bu), 7.65 (d, J = 8.6 Hz, 2H, Ar−H), 7.70 (d, J = 8.6 Hz, 4H, Ar−H), 7.74 (d, J = 8.6 Hz, 2H, Ar−H), 7.75 (d, J = 8.6 Hz, 2H, Ar−H), 7.89 (d, J = 8.6 Hz, 4H, Ar−H), 7.95 (d, J = 8.6 Hz, 2H, Ar−H), 7.87, 7.88, 7.89 (d, J = 8.6 Hz, 6H, Ar−H), 8.45 (m, 4H, Leu-NH), 9.37 (s, 1H, tBuCONH), 10.25 (s, 1H, t-BuCONHC6H4CO-Leu-NHC6H4), 10.31 (s, 3H, NHC6H4). ESI-MS Calcd for C68H88N10O10 ([M + Na]+): 1227.7; found 1227.5, ([M − H]−): 1203.7; found 1203.5. Boc-NHC6H4COOH. p-Aminobenzoic acid (8.0 g, 0.058 mol) was dissolved in a mixture of 1,4-dioxane (80 mL), water (40 mL), and 1 M NaOH(aq) (60 mL). To the solution was added (Boc)2O (14.0 g, 0.064 mol) cooling in an ice bath. The reaction was monitored by TLC. After stirring for 10 h at room temperature, to the solution was added (Boc)2O (10.0 g, 0.046 mol) at 0 °C again and stirred overnight. Unreacted amino acid remained; thus, additional (Boc)2O (2.0 g, 9.2 mmol) was added every 9 h until the spot of the reactant was almost vanished. The solution was concentrated under reduced pressure. To the residue was added water and ethyl acetate. The aqueous layer was acidified with citric acid and extracted with ethyl acetate. Organic layer was washed with saturated NaCl(aq), dried over Na2SO4, and concentrated under reduced pressure. An obtained powder was collected with filtration, washed with ethyl acetate, and dried over P2O5 in vacuo. Yield 12.0 g (87% based on aminobenzoic acid). 1H NMR (DMSO-d6): δ 1.48 (s, 9H, Boc), 7.54 (d, J = 8.8 Hz, 2H, Ar−H), 7.82 (d, J = 8.8 Hz, 2H, Ar−H), 9.68 (s, 1H, Boc-NH), 12.54 (s, 1H, COOH). Boc-NHC6H4COOPac. To a DMF solution (25 mL) of BocNHC6H4COOH (11.4 g, 0.048 mol) was added NEt3 (6.8 mL, 0.048 mol) in ice bath. The solution became turbid. Addition of DMF (10 mL) gave a clear solution. Phenacyl bromide (9.6 g, 0.048 mol) was added to the mixture, keeping the temperature. After stirring overnight, to the mixture were added DMF (20 mL), NEt3 (0.4 mL, 0.0028 mol), and phenacyl bromide (3.9 g, 0.020 mol). The mixture was stirred overnight, and heating from 30 to 90 °C for 6 h to dissolved precipitate. The precipitate could not be soluble and filtered out. The filtrate was concentrated under reduced pressure. Addition of water and ethyl acetate gave a yellow powder and yellow supernatant. This powder was collected with filtration and washed successively with water, 4% NaHCO3(aq), water, 10% citric acid(aq), and water and recrystallized from hot ethyl acetate, collected with filtration, washed with ethyl acetate, and dried over P2O5 in vacuo. The supernatant was washed successively with 4% NaHCO3(aq), saturated NaCl(aq), 10% citric acid(aq), and saturated NaCl(aq), dried over Na2SO4, and concentrated in vacuo. The solution was evaporated. An obtained powder was recrystallized from hot ethyl acetate, collected with filtration, washed with ethyl acetate, and dried over P2O5 in vacuo. Yield 11.5 g (67%). 1H NMR (DMSO-d6): δ 1.49 (s, 9H, Boc), 5.68 (s, 2H, CH2(OPac)), 7.57 (m, 2H, m-H(OPac)), 7.61 (d, J = 8.8 Hz, 2H, Ar−H), 7.70 (m, 1H, p-H(OPac)), 7.92 (d, J = 8.8 Hz, 2H, Ar− H), 8.00 (m, 2H, o-H(OPac)), 8.80 (s, 1H, Boc-NH). HCl·NH2C6H4COOPac. Boc-NHC6H4COOPac (6.7 g, 0.20 mol) was suspended in saturated HCl−ethyl acetate solution (150 mL). After stirring overnight, the solvents were removed under reduced pressure. The obtained powder was collected with filtration and washed with ethyl acetate. Yield 5.2 g (94%). 1H NMR (DMSO-d6): δ 5.90 (s, 2H, CH2(OPac)), 6.93 (d, J = 8.3 Hz, 2H, Ar−H), 7.87 (m, 2H, m-H(OPac)), 8.00 (m, 1H, p-H(OPac)), 8.02 (d, J = 8.3 Hz, 2H, Ar−H), 8.30 (m, 2H, o-H(OPac)). Boc-Leu-NHC6H4COOPac. NH2C6H4COOPac was prepared by a neutralization of HCl·NH2C6H4COOPac (4.7 g, 0.018 mol) using triethylamine in ethyl acetate. NH2C6H4COOPac, Boc-Leu-OH·H2O (4.5 g, 0.018 mol) and HOBt (3.0 g, 0.022 mol) were dissolved in DMF (30 mL). To the solution was added DCC (4.8 g, 0.023 mol) cooling in an ice bath. The solution was stirred at this temperature for 1.5 h and at room temperature overnight. After removal of solvents under reduced pressure, the residue was dissolved in ethyl acetate. The solution was washed with water, 4% NaHCO3(aq), water, 10% citric acid(aq), and water and then concentrated to dryness in vacuo. Yield G

DOI: 10.1021/acs.macromol.7b00718 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules

mL) at −25 °C. The solution was stirred for 2 h keeping the temperature and at room temperature overnight. The solvents were removed under reduced pressure. The residue was washed with water, 4% NaHCO3(aq), water, 2% HCl(aq), and water. The residue was reprecipitated with ethanol, collected with filtration, and dried in vacuo. Yield 1.14 g (82%). 1H NMR (DMSO-d6): δ 0.93 (m, 24H, Leu-δ(CH3)2), 1.23 (s, 9H, t-BuCO), 1.57−1.85 (m, 12H, Leu-β-CH2, γCH), 4.65 (br, 4H, (C6H4CO−)Leu-α-H), 5.70 (s, 2H, CH2(OPac)), 7.57 (m, 2H, m-H(OPac)), 7.67−7.74 (m, 9H, Ar−H, p-H(OPac)), 7.81 (d, J = 8.9 Hz, 2H, Ar−H), 7.83−7.90 (d, 8H, Ar−H), 7.98 (d, J = 8.9 Hz, 2H, Ar−H), 8.00 (m, 2H, o-H(OPac)), 8.47 (m, 4H, LeuNH), 9.37 (s, 1H, t-BuCONHC6H4), 10.25 (s, 1H, t-BuCONHC6H4COLeu-NHC6H4), 10.31, 10.48 (s, 3H, C6H4CO-Leu-NHC6H4). t-BuCONHC 6 H 4 CO-(Leu-NHC 6 H 4 CO) 4 OH (14). t-BuCONHC 6 H 4 CO-(Leu-NHC6 H 4 CO) 4 OPac (1.05 g, 0.83 mmol) was suspended in acetic acid (160 mL) and DMF (4 mL). To the mixture was added zinc powder (1.62 g, 25 mmol). The mixture was heated and stirring overnight at 45 °C for 3 days. The precipitate was filtered out, and the filtrate was concentrated to dryness under reduced pressure. The residue was washed with water and THF. Yield 0.81 g (85%). 1H NMR (DMSO-d6): δ 0.93 (m, 24H, Leu-δ-(CH3)2), 1.23 (s, 9H, t-BuCO), 1.56−1.77, (m, 12H, Leu-β-CH2, γ-CH), 4.65 (br, 4H, Leu-α-CH), 7.63 (d, J = 8.8 Hz, 2H, Ar−H), 7.70 (d, J = 8.8 Hz, 4H, Ar−H), 7.74 (d, J = 8.8 Hz, 2H, Ar−H), 7.83−7.94 (m, 12H, Ar− H), 8.46 (m, 4H, Leu-NH), 9.37 (s, 1H, t-BuCONHC6H4), 10.23 (s, 1H, t-BuCONHC6H4COLeu-NHC6H4), 10.31 (s, 3H, NHC6H4). t-BuCONHC6H4CO-(Leu-NHC6H4CO)8-NH-t-Bu (4). Compounds 13 (93 mg, 0.089 mmol), 14 (103 mg, 0.89 mmol), and HOBt (15 mg, 0.11 mmol) were dissolved in DMF (2.0 mL). To the solution were added NEt3 (0.01 mL, 0.089 mmol) in an ice bath and WSCD (0.02 mL, 0.11 mmol) at −25 °C. The solution was stirred for 2 h keeping the temperature and at room temperature overnight. The solvents were removed under reduced pressure. The residue was washed successively with water, 4% NaHCO3(aq), water, 2% HCl(aq), water and with ethanol and dried in vacuo. The crude product was purified by repeated reprecipitation from DMF/THF. Yield 113 mg (59%). 1H NMR (DMSO-d6): δ 0.93 (m, 48H, Leu-δ-(CH3)2), 1.23 (s, 9H, tBuCO), 1.36 (s, 9H, N-t-Bu), 1.56−1.80, (m, 24H, Leu-β-CH2, γCH), 4.45, 4.65 (br, 8H, Leu-α-CH), 7.56 (s, 1H, NH-t-Bu), 7.65 (d, J = 8.6 Hz, 2H, Ar−H), 7.70 (d, J = 8.6 Hz, 14H, Ar−H), 7.74 (d, J = 8.6 Hz, 2H, Ar−H), 7.89 (d, J = 8.6 Hz, 14H, Ar−H), 8.45 (m, 8H, Leu-NH), 9.37 (s, 1H, t-BuCONHC6H4), 10.25, 10.31 (s, 8H, NHC6H4). t-BuCONHC6H4CO-(Leu-NHC6H4CO)n-NH-t-Bu (Polymer 5). BocLeu-NHC6H4COOH (680 mg, 1.9 mmol) was dissolved saturated HCl−ethyl acetate solution (24 mL) in an ice bath. After stirring overnight at room temperature, solvents were removed under reduced pressure. The residue was washed with diethyl ether and dried under reduced pressure to give deprotected monomer, HCl·H-LeuNHC6H4COOH (500 mg) in 90% yield. This monomer (360 mg, 1.3 mmol), NEt3 (0.18 mL, 1.3 mmol), and HOBt (203 mg, 1.5 mmol) were dissolved in DMF (7 mL). To the solution was added WSCD (0.27 mL, 1.5 mmol) in an ice bath ands stirred at room temperature. The solvents were removed under reduced pressure. The residue was washed with water and dried over P2O5 under reduced pressure to give a power (358 mg). The obtained H-(LeuNHC6H4CO)n-OH (110 mg) and HOBt (71 mg, 0.52 mmol) were dissolved in DMF (3 mL). To the solution were added tert-butylamine (0.47 mL, 0.44 mmol) in an ice bath and WSCD (0.1 mL, 0.5 mmol) at −25 °C. After stirring for 2 h at −25 °C and overnight at room temperature, the solution was concentrated to dryness under reduced pressure. The residue was washed with water, 4% NaHCO3(aq), water, and diethyl ether and dried under reduced pressure over P2O5 to give H-(Leu-NHC6H4CO)n-NH-t-Bu. On the other hand, t-BuCONHC6H4COOH (50 mg, 0.23 mmol) was suspended in SOCl2 (4 mL). The mixture was stirred and became clear after 5 h. The solvents were removed under reduced pressure to give t-BuCONHC6H4COCl in quantitative yield. The acid chloride and H-(Leu-NHC6H4CO)n-NH-tBu (60 mg) were dissolved in N,N-dimethylacetamide (DMA, 0.5 mL). The solution was stirred for 2 days and filtrated. After removal of

3.7 g (44%). 1H NMR (DMSO-d6): δ 0.88 (d, 3H, J = 1.5 Hz, Leu-δ(CH3)2), 0.91 (d, 3H, J = 1.5 Hz, Leu-δ-(CH3)2), 1.38 (s, 9H, Boc), 1.43−1.65 (m, 3H, Leu-β-CH2, γ-CH), 4.14 (br, 1H, Leu-α-CH), 5.70 (s, 2H, CH2(OPac)), 7.09 (d, 1H, J = 8.8 Hz, Leu-NH), 7.57 (m, 2H, m-H(OPac)), 7.70 (m, 1H, p-H(OPac)), 7.78 (d, J = 8.9 Hz, 2H, Ar− H), 7.98 (d, J = 8.9 Hz, 2H, Ar−H), 8.00 (m, 2H, o-H(OPac)), 10.31 (s, 1H, NHC6H4). ESI-MS: m/z; 491.1 [M + Na+] (calcd 491.2), 466.9 [M − H+] (calcd 467.2). HCl·H-Leu-NHC6H4COOPac. This compound was synthesized from Boc-Leu-NHC6H4COOPac by a similar method as described for HCl· NH2C6H4COOPac. Yield 2.7 g (84%). 1H NMR (DMSO-d6): δ 0.94 (d, 6H, Leu-δ-(CH3)2), 1.70 (m, 3H, Leu-β-CH2, γ-CH), 3.98 (br, 1H, Leu-α-CH), 5.72 (s, 2H, CH2(OPac)), 7.09 (d, 1H, J = 8.8 Hz, LeuNH), 7.58 (m, 2H, m-H(OPac)), 7.71 (m, 1H, p-H(OPac)), 7.80 (d, J = 8.6 Hz, 2H, Ar−H), 8.00 (m, 2H, o-H(OPac)), 8.05 (d, J = 8.6 Hz, 2H, Ar−H), 8.26 (br, 3H, Leu-NH3Cl), 10.33 (br, 1H, NHC6H4). Boc-(Leu-NHC6H4CO)2OPac. This compound was synthesized from 8 and HCl·H-Leu-NHC6H4COOPac by a similar method as described for Boc-Leu-NH2C6H4COOPac. Yield, 2.6 g (48%). 1H NMR (DMSO-d6): δ 0.92 (m, 12H, Leu-δ-(CH3)2), 1.37 (s, 9H, Boc), 1.43−1.81 (m, 6H, Leu-β-CH2, γ-CH), 4.13 (br, 1H, Leu-α-CH), 4.65 (br, 1H, (C6H4CO−)Leu-α-H), 5.70 (s, 2H, CH2(OPac)), 7.03 (d, 1H, J = 7.8 Hz, (Boc-)Leu-NH), 7.57 (m, 2H, m-H(OPac)), 7.68 (d, J = 8.7 Hz, 2H, Ar−H), 7.70 (m, 1H, p-H(OPac)), 7.81 (d, J = 8.8 Hz, 2H, Ar−H), 7.88 (d, J = 8.7 Hz, 2H, Ar−H), 7.99 (d, J = 8.8 Hz, 2H, Ar−H), 8.00 (m, 2H, o-H(OPac)), 8.49 (d, 1H, J = 7.6 Hz, (C6H4CO−)Leu-NH), 10.14 (s, 1H, Boc-Leu-NHC6H4), 10.48 (s, 1H, C6H4CO-Leu-NHC6H4). ESI-MS: m/z; 723.2 [M + Na+] (calcd 723.3), 699.1 [M − H+] (calcd 699.3). HCl·H-(Leu-NHC6H4CO)2OPac. Boc-(LeuNHC6H4CO)2OPac (1.1 g, 0.0015 mol) was dissolved in TFA (1.7 mL) and CHCl2 (1.7 mL) in an ice bath. After stirring at room temperature for 1 h, the solution was removed under reduced pressure. To the residual oil was added saturated HCl−ethyl acetate solution in an ice bath. Then the solvents were evaporated. This operation was repeated three times. The obtained powder was washed with ether. Yield, 0.95 g (97%). 1H NMR (DMSO-d6): δ 0.94 (m, 12H, Leu-δ-(CH3)2), 1.56−1.83 (m, 6H, Leu-β-CH2, γ-CH), 3.97 (br, 1H, Leu-α-CH), 4.68 (br, 1H, (C6H4CO−)Leu-α-H), 5.70 (s, 2H, CH2(OPac)), 7.57 (m, 2H, mH(OPac)), 7.70 (d, J = 8.9 Hz, 2H, Ar−H, m, 1H, p-H(OPac)), 7.81 (d, J = 8.9 Hz, 2H, Ar−H), 7.95 (d, J = 8.9 Hz, 2H, Ar−H), 7.99 (d, J = 8.8 Hz, 2H, Ar−H), 8.00 (m, 2H, o-H(OPac)), 8.27 (br, 3H, LeuNH3Cl), 8.58 (d, 1H, J = 7.8 Hz, (C6H4CO−)Leu-NH), 10.53, 10.78 (s, 2H, NHC6H4). Boc-(LeuNHC6H4CO)4OPac. This compound was synthesized from 10 and HCl·H-(Leu-NHC6H4CO)2OPac by a similar method as described for Boc-Leu-NH2C6H4COOPac. Yield, 1.6 g (82%). 1H NMR (DMSO-d6): δ 0.92 (m, 24H, Leu-δ-(CH3)2), 1.37 (s, 9H, Boc), 1.57−1.85 (m, 12H, Leu-β-CH2, γ-CH), 4.13 (br, 1H, Leu-α-CH), 4.65 (br, 3H, (C6H4CO−)Leu-α-H), 5.70 (s, 2H, CH2(OPac)), 7.02 (d, 1H, J = 8.2 Hz, (Boc-)Leu-NH), 7.57 (m, 2H, m-H(OPac)), 7.61, 7.78 (d, 6H, Ar−H), 7.70 (m, 1H, p-H(OPac)), 7.80 (d, J = 8.8 Hz, 2H, Ar−H), 7.88 (d, 6H, Ar−H), 7.98 (d, J = 8.9 Hz, 2H, Ar−H), 8.00 (m, 2H, o-H(OPac)), 8.47 (m, 3H, (C6H4CO−)Leu-NH), 10.14 (s, 1H, Boc-Leu-NHC6H4), 10.31, 10.48 (s, 3H, C6H4CO-Leu-NHC6H4). HCl·H-(LeuNHC6H4CO)4OPac. This compound was synthesized by the similar method as described for HCl·H-(LeuNHC6H4CO−)2OPac. Yield, 0.60 g (90%). 1H NMR (DMSO-d6): δ 0.93 (m, 24H, Leu-δ-(CH3)2), 1.57−1.85 (m, 12H, Leu-β-CH2, γCH), 3.97 (br, 1H, Leu-α-CH), 4.66 (br, 3H, (C6H4CO−)Leu-α-H), 5.67 (s, 2H, CH2(OPac)), 7.57 (m, 2H, m-H(OPac)), 7.67−7.74 (m, 7H, Ar−H, p-H(OPac)), 7.81 (d, J = 8.9 Hz, 2H, Ar−H), 7.89 (d, 4H, Ar−H), 7.94 (d, J = 8.7 Hz, 2H, Ar−H), 7.98 (d, J = 8.7 Hz, 2H, Ar− H), 8.00 (m, 2H, o-H(OPac)), 8.28 (br, 3H, Leu-NH3Cl), 8.50 (m, 3H, (C6H4CO−)Leu-NH), 10.34, 10.36, 10.50, 10.81 (s, 4H, NHC6H4). t-BuCONHC 6 H 4 CO-(Leu-NHC 6 H 4 CO) 4 OPac. HCl·H-(LeuNHC6H4CO)4OPac (1.2 g, 1.1 mmol), 6 (0.24 g, 1.1 mmol), and HOBt (0.18 g, 1.3 mmol) was dissolved in DMF (4 mL). To the solution were added NEt3 (0.15 mL) in an ice bath and WSCD (0.24 H

DOI: 10.1021/acs.macromol.7b00718 Macromolecules XXXX, XXX, XXX−XXX

Macromolecules

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solvents under reduced pressure, the residue was washed with water, 2% HCl(aq), and water. The residue was washed with ethyl acetate and dried in vacuo to give a crude product (55 mg), which was reprecipitated from DMF and THF several times. The precipitate was collected with filtration, washed with THF, and dried in vacuo. Yield, 12 mg. 1H NMR (DMSO-d6): δ 0.93 (m, Leu-δ-(CH3)2), 1.22 (s, 9H, t-BuCO), 1.36 (s, 9H, N-t-Bu), 1.56−1.80, (m, Leu-β-CH2, γ-CH), 4.65 (br, Leu-α-CH), 7.55 (br, NH-t-Bu), 7.70 (d, Ar−H), 7.88 (d, Ar−H), 8.46 (m, Leu-NH), 9.32 (s, 1H, t-BuCONHC6H4), 10.30 (s, NHC6H4). Physical Measurements. UV spectra were recorded on a Shimadzu UV-3100PC spectrophotometer in 0.1 mM dimethyl sulfoxide (DMSO) solution using 1 mm path length at 303 K. CD spectra were recorded on a Jasco J-820w spectropolarimeter, and samples were prepared as DMSO solution using a 0.1 mm path length cell at 303 K. 1H NMR spectra were recorded on JEOL JNM-EX270 and JNM-LA500 spectrometers in DMSO-d6 at 30 °C. Nuclear Overhauser effect (NOE) correlated spectroscopy (NOESY) spectra were recorded on a Varian UNITYplus 600 MHz spectrometer in DMSO-d6 at 30 °C. ESI-MS experiments were performed on a Finnigan MAT LCQ ion trap mass spectrometer in a methanol solution. The molecular weight of polymer was determined by SEC on systems equipped with two-column sets (TSKgel α-M 7.8 mm × 300 mm, beads size = 13 μm) and UV detector (Shimadzu) at 40 °C using DMF with 0.1 M LiBr as an eluent at a flow rate of 0.5 mL/min. Structure Determination of the Polymer 5. Complete assignments of peaks were made by total correlation spectroscopy (TOCSY), double quantum filtered correlation spectroscopy (DQFCOSY), and NOESY measurements. Virtual model CH3CONHC6H4CO-Leu-NHC6H4CONHCH3 (1′) was used for the calculation to determine the local conformation of leucine residue. A simulated annealing (SA) protocol was used to this model using NMR constraints (15 NOEs and 2 3J coupling constants of 3), where the vicinal coupling constants 3JHα‑NH and 3JHα−βH are 7.6 and 6.4 Hz, respectively. Fifty structures were generated and optimized. The obtained 10 lowest energy structures showed satisfied agreement of backbone (RMSD = 0.0023). The lowest energy structure of leucine moiety was applied to the energy minimization of polymer 5′. The minimized structure was connected to reconstruct the complete chemical structure of polymer 5′. The leucine residue and the paminobenzoic acid part are treated as rigid bodies. Two dihedral angles (ϕ, ψ) were fixed during the calculations. Free rotations about the phenylene groups were assumed. Energy minimization was performed using the consistent valence force field (CVFF).43 Theoretical Calculations. Geometry optimizations were performed using Becke’s three-parameter hybrid functionals (B3LYP) in the Gaussian 0944 program package. The 6-31G** basis set was employed. The initial model 1′ was made from analogous crystal structures with some modifications. The terminal tert-butyl group was replaced by a methyl group. The optimization in a vacuum and CHCl3 resulted in unsuitable structures with intramolecular short NH···OC contacts. The geometrical optimization of 1′ in DMSO using the polarizable continuum model (PCM) as self-consistent reaction field (SCRF) method resulted in a reasonable structure. The initial model 2′ was constructed from the optimized structure of 1′ as a partial structure on the software ChemBio3D (ver. 13.0.2.3021, Cambridge Soft). The initial model and the geometrical optimization of 3′ were performed in a similar manner for 2′. The absorption spectra were simulated by time-dependent (TD) DFT calculations in DMSO after the geometrical optimization. The proposed structure of 5′ shown in Figure 7 was made from the optimized structure of 3′ by combination of five molecules of 3′ as a rigid body with deletion of overlapped terminal groups and one residue. The molecules were manipulated carefully to avoid deformation on the software.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.7b00718. Total synthetic scheme, determination of helicity, plots of Δε against T, NOESY spectrum, MOs, and calculated absorbance (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (T.O.). ORCID

Taka-aki Okamura: 0000-0002-9005-4015 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by JSPS KAKENHI Grants JP20550063 and JP 26410072. REFERENCES

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DOI: 10.1021/acs.macromol.7b00718 Macromolecules XXXX, XXX, XXX−XXX